Chitosan is an amino-polysaccharide derived from natural sources. It is obtained by deacetylation of chitin, which is typically contained in crustacean animals such as crabs and crayfish, insects and fungi. Chitosan has in vivo indigestible antigenicity indigestible in vivo and is useful in medical applications, for instance, as an inhibitor against the growth of tumors utilizing its macrophage activating properties and a caries-preventing agent utilizing its antimicrobial properties as well as in water treatment as a chelating agent for capturing heavy metal ions and as a cationic flocculant, enzyme immobilizing carriers and in cosmetics utilizing its good viscosity stabilizing effect, water-retentivity and lustering properties.
Sulfonated chitosan has a structure similar to that of heparin which is a typical anticoagulant and has been studied since the most earliest time among various types of chitosan derivatives due to its potentiality for use as an inexpensive anticoagulant. The relation between the structure and the activity thereof is not yet clear but it is naturally expected that the physiological activity of sulfonated chitosan will be influenced by sulfonated positions and, in fact, it is known that some sulfonated chitosans exhibit no activity, in which sulfon-groups are introduced on amino groups. The sulfon-group introduced on the amino group should be then selectively eliminated. Furthermore, when sulfonated chitosan is used utilizing its properties as a water-soluble amphoteric polymer, for instance, as a paper strength improver, flocculant, separating agent, and for immunogen carriers, water-soluble drug carriers and the like, amino-groups thereof must be free.
There are not a few reports on the sulfonation of chitosan. The most typical process among those reported comprises treating chitosan either with chlorosulfonic acid (ClHSO.sub.3) in pyridine or with sulfur trioxide (SO.sub.3) in dimethylformamide (DMF). Both the hydroxyl and amino groups are, however, sulfonated according to these processes (See M. L. Wolfrom, T. M. Shen Han : JACS 81,1764(1959)). Selective sulfonation of amino groups can be carried out by using pyridine/sulfur trioxide (D. T. Warner, L. L. Coleman: J. Org. Chem., 23, 1133(1958)). Russian chemists recently reported that the hydroxyl group is selectively reacted by 2-propanol/sulfuric acid or DMF/chlorosulfonic acid (L. I. Batura, G. A. Vilkhoreva, R. Noreika, L. S. Gal'braikh, Z. A. Regovin: Cell. Chem. Tech., 15, 487 (1981)). This is, however, a laboratory process and not directly applicable to commercial production since expensive chlorosulfonic acid is used therein.
The conventional process for producing sulfonated chitosan comprises dissolving pretreated chitosan in a DMF solution containing 5-10 molar equivalent of a sulfonating agent (SO.sub.3 -DMF complex), carrying out the sulfonating reaction under agitation for not less than 12 hours, adding a large amount of alcohol and a large amount of alkaline aqueous solution to precipitate the resulting sulfonated chitosan in which amino groups are sulfonated as well, Na.sub.2 SO.sub.4 by-product and unreacted chitosan, filtering the mixture to separate a solution containing DMF, alcohol and water and a deposit containing the sulfonated chitosan, Na.sub.2 SO.sub.4 and unreacted chitosan. The deposit is washed with a large amount of alcohol, dissolved in water and filtered. The Na.sub.2 SO.sub.4 accompanying from the filtrate is removed by dialysis and after further purification treatments, sulfonated chitosan is finally obtained. The thus obtained chitosan contains, however, sulfonated C2-amino groups and should be subjected to a desulfonation treatment which comprises dissolving the sulfonated chitosan in a methanol/DMSO solution at 50.degree. C. for about 1.5 hours under agitation, diluting the solution with water, dialyzing the diluted solution for 3-4 days after neutralization with an alkaline aqueous solution. Concentrating and lyophilizing steps follows and then the desired chitosan in which only hydroxyl groups are sulfonated is obtained.
The above-described conventional process is disadvantageous in an economical point of view since it requires 5-10 times as much as the theoretical amount of sulfonating agent and a large amount of solvent in the sulfonating reaction step and it also requires a neutralization step with alkali and addition of a large amount of alcohol to precipitate the dissolving sulfonated chitosan after the reaction step. Furthermore, since the sodium sulfate precipitated in a large amount along with the object compound in the neutralization step is viscous in DMF, filtration thereof is extremely troublesome and a further separation step such as dialysis is necessary in order to separate the object compound from the sodium sulfate.
The conventional process comprises many steps including a desulfonating step and is not satisfactory for practice on a commercial scale.
The object of the present invention is to provide an economical process for preparing a sulfonated chitosan with C3/C6 hydroxyl groups thereof selectively sulfonated wherein the selective sulfonation of hydroxyl groups of chitosan is achieved by an inexpensive sulfonating agent and the unreacted chitosan and the used solvent can be recycled without any refining treatment such as fractional distillation.
We conducted a research in order to solve the above-described problems and found reaction conditions that SO.sub.3 -DMF complex or ClHSO.sub.3 -DMF complex is suitable as a sulfonating agent, selected solvents which dissolve the sulfonating complexes but do not dissolve the object compounds and found pretreatment conditions and a solvent system which enables the sulfonation to proceed like a reaction in a homogeneous phase though it is, in fact, substantially a heterogeneous phase as well as quaternizing treatment conditions and a system therefor by which amino groups of chitosan are protected from sulfonation.